Optically active D-.alpha.-amino acids are important compounds as intermediates of medicaments, and in particular, D-phenylglycine, D-parahydroxyphenylglycine and the like, which are intermediates for the production of semi-synthetic penicillins and semisynthetic sepharospolines, are useful compounds from an industrial point of view. As the production process for such D-.alpha.-amino acids, a process is known in which these compounds are obtained by the removal of carbamoyl groups of the corresponding D-N-carbamoyl-.alpha.-amino acids, wherein the removal of carbamoyl groups is effected by a chemical method (the specification of the Japanese Patent Publication No. 58-4707) or by the utilization of enzyme reactions of microorganisms (the specifications of the Japanese Patent Publication Nos. 57-18793, 63-20520 and 1-48758, and the Japanese Patent Application No. 2-407922).
However, because a mineral acid such as sulfuric acid is used at great amounts in the chemical method employed for the removal of carbamoyl groups, serious problems on the environment may occur in relation to the treatment thereof and the like. On the other hand, the method utilizing an enzyme reaction has disadvantages that the amount of enzyme produced is not satisfactory and there are some difficulties in its properties even if mass production is made possible, so that enzymes with reactivity to substrates and enzyme stability have not yet been found.
In general, some enzymes have poor stability, and when such an enzyme is prepared, a stabilizing agent is added or a means of preventing inactivation such as treatment at low temperatures is employed. When an enzyme is used in an actual reaction at ordinary or high temperatures, attention will be directed to the stability of the enzyme. In particular case where an enzyme is used on an industrial scale, its stability often has an effect upon the product cost. Also, in particular case where an enzyme is used on an industrial scale, as a means of allowing the enzyme reaction to proceed with advantage, the enzyme is repeatedly used in the reaction as a so-called bioreactor such as an immobilized enzyme or immobilized bacterial cells, and even at this time, the stability of the enzyme will give a limitation to the number of use and have an important effect upon the product cost.
In the present invention, attention is paid to the thermostability as an index of the enzyme stability and an improvement in the thermostability of an enzyme is achieved using a genetic engineering means, whereby the stability of this enzyme is increased and this makes it possible to produce a stabilized enzyme having excellent advantages for industrial applications.
To solve such a problem, the present invention has an object to prepare a decarbamylase with high reactivity to D-N-carbamoyl-.alpha.-amino acids as the substrate and excellent stability by an improvement of currently available decarbamylases and to produce D-.alpha.-amino acids using this enzyme with high efficiency.